Gene which regulates the dormancy of wheat seed

ABSTRACT

The present invention provides an isolated gene which regulates the dormancy of wheat seed, an antisense DNA to said gene, an isolated protein having activity which alters the degree of dormancy of wheat seed, a recombinant vector containing the gene, a transformant containing the vector; and a method for producing the protein.

FIELD OF THE INVENTION

[0001] The present invention relates to an isolated gene which regulates the dormancy (resistance to preharvest sprouting) of wheat seed, an antisense DNA to said gene, an isolated protein having activity which alters the degree of wheat seed dormancy, a recombinant vector containing the gene, a transformant containing the vector, and a method for producing the protein.

BACKGROUND OF THE INVENTION

[0002] VP1 has been isolated so far as one gene which is a cause of Viviporus of corn, and elucidated to be a transcriptional regulatory gene essential for formation of seed dormancy (McCarty et al., 1991). Furthermore, ABI3 gene isolated around the same time as a causal gene of abscisic acid-insensitive mutation of Arabidopsis seed, has been revealed to be a homologous gene of VP1 (Giraudat et al., 1992). Based on these studies, VP1 gene has been found to be a transcriptional factor which acts in the abscisic acid signaling system of a seed, playing a key role in formation of seed dormancy.

[0003] As seen from the above, at least one gene which plays a key role in the formation of seed dormancy is known to exist in some kinds of plants, however, it is still an open question as to what kind of gene action causes differences in the degree of wheat seed dormancy.

SUMMARY OF THE INVENTION

[0004] The objectives of the present invention are to elucidate what kind of gene action causes differences in the degree of the wheat seed dormancy, and to provide a gene which regulates dormancy.

[0005] The inventors of the present invention have studied extensively and intensively to solve the above problems and have now found that the expression level of a certain gene exhibits correlation with the degree of dormancy in seeds of wheat varieties having different degrees of dormancy, and in wheat seeds in which dormancy has been disrupted by long-term storage. The present invention has been led to completion based on the above findings.

[0006] (i) Accordingly, the present invention relates to an isolated gene encoding a protein selected from the group consisting of the following (a) and (b):

[0007] (a) a protein comprising the amino acid sequence of SEQ ID NO: 2;

[0008] (b) a protein which comprises the amino acid sequence having deletions, substitutions or additions of one a plurality of amino acids in the amino acid sequence of SEQ ID NO: 2, and has activity which alters the degree of dormancy of wheat seed.

[0009] (ii) Furthermore, the invention relates to an antisense DNA to the gene of (i).

[0010] (iii) Still further, the present invention relates to an isolated protein selected from the group consisting of the following (a) and (b):

[0011] (a) a protein comprising the amino acid sequence of SEQ ID NO: 2;

[0012] (b) a protein which comprises the amino acid sequence having deletions, substitutions or additions of one or a plurality of amino acids in the amino acid sequence of SEQ ID NO: 2, and has activity which alters the degree of dormancy of wheat seed.

[0013] (iv) In addition, the invention relates to a recombinant vector comprising the gene of (i).

[0014] (v) The invention also relates to a recombinant vector comprising the antisense DNA of

[0015] (vi) Further, the invention relates to a transformant comprising the recombinant vector of (iv).

[0016] (vii) Further, the invention relates to a transformant comprising the recombinant vector of (v).

[0017] (viii) The invention also relates to a method for producing a protein having activity which alters the degree of dormancy of wheat seed comprising culturing the transformant of (vi) and recovering the protein from the resultant culture.

[0018] (ix) Further, the invention relates to a method for producing a protein having activity which alters the degree of dormancy of wheat seed comprising culturing the transformant of (vii) and recovering the protein from the resultant culture.

[0019] This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No.2000-350363, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 shows the results of comparison between a putative amino acid sequence encoded by a gene of the invention (SEQ ID NO: 2) and a putative amino acid sequence encoded by VP1 gene which is known in relation to different types of plants. Herein, Ta VP1 shows a putative amino acid sequence from a sequence of a gene of the invention presumed to be wheat VP1 gene, and AfVP1, Os and ZmVP1 show putative amino acid sequences from a sequence of VP1 gene which is known in relation to cat grass, rice and field corn, respectively

[0021]FIG. 2 shows the results obtained by performing Northern blot using DNA of a gene of the invention as a probe on mRNA from a seed of wheat varieties having various degrees of the dormancy and abscisic acid-sensitivity.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will be described in detail below.

[0023] An isolated gene of the present invention is characterized in that it encodes a protein selected from the group consisting of the following (a) and (b):

[0024] (a) a protein comprising the amino acid sequence of SEQ ID NO: 2;

[0025] (b) a protein which comprises the amino acid sequence having deletions, substitutions or additions of one or a plurality of amino acids in the amino acid sequence of SEQ ID NO: 2 and has activity which alters the degree of dormancy of wheat seed.

[0026] An isolated gene which has the nucleotide sequence of SEQ ID NO: 1 can be exemplified as a gene of the invention.

[0027] Among the above-mentioned genes, an gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 can be obtained, for example, from a wheat according to a method which will be described in Example 1 hereinafter

[0028] Further, as shown in SEQ ID NO: 1, a gene in which the nucleotide sequence is once determined can be obtained, for example, by designing and synthesizing appropriate primers based on these nucleotide sequences, thereby performing PCR.

[0029] An isolated gene encoding a protein which comprises the amino acid sequence having deletions, substitutions or additions of one or a plurality of amino acids in the amino acid sequence of SEQ ID NO: 2 can be obtained according to common techniques used at the time when this application is filed, for example, site-directed mutagenesis method (Zoller et al., Nucleic Acids Res. 10 6478-6500, 1982) by means of modifying a sequence of a gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 2. In this connection, “one or a plurality of amino acids” used herein is normally one or more amino acids, but is not limited as long as activity which alters the degree of dormancy of wheat seed is maintained in the protein where an amino acid sequence is modified by the above-mentioned method.

[0030] Furthermore, “a protein having activity which alters the degree of dormancy of wheat seed” means a protein which enhances dormancy (i.e. properties which prevent sprouting) by enhancing (improving) the resistance to dehydration in a seed where said protein is produced in the embryo thereof.

[0031] Furthermore, the term “isolated” used herein, means altered “by the hand of man” from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.

[0032] By introducing a gene of the invention into varieties of wheat to regulate the expression level thereof, the degree of abscisic acid-sensitivity of seeds in those varieties can be altered, thereby the degree of dormancy of said seed can be changed. Herein, introduction of a gene can be carried out according to a method known to those skilled in the art for example, by introducing a gene of the invention into a protoplast of a plant from the wheat variety of interest, and reproducing.

[0033] Still further, an antisense DNA of the invention is characterized to be the antisense DNA to the above-described gene.

[0034] “Antisense DNA” used herein means DNA where the expression thereof functions to inhibit the expression of the above-mentioned gene, for example, DNA or fragment thereof which is complementary to the sequence of the gene in the above. Moreover, as long as it has the inhibiting function described above, the antisense DNA of the invention is not limited to one which is completely complementary to the above-mentioned gene, but can include, for example, partially mismatched sequence.

[0035] Accordingly, introducing the antisense DNA of the invention into various wheat varieties to inhibit the expression of a gene of the invention therein enables regulation of the dormancy of tie seed,

[0036] An isolated protein of the invention is characterized to be selected from the group consisting of the following (a) and (b):

[0037] (a) a protein comprising the amino acid sequence of SEQ ID NO: 2;

[0038] (b) a protein which comprises the amino acid sequence having deletions, substitutions or additions of one or a plurality of amino acids in the amino acid sequence of SEQ ID NO: 2, and has activity which alters the degree of dormancy of wheat seed.

[0039] Among proteins of the invention, a protein shown in an amino acid sequence of SEQ ID NO: 2 can be obtained, for example, by converting each series of 3 bases in the nucleotide sequence of SEQ ID NO: 1 by one amino acid which is encoded (or determined) by the combination of the bases (i.e. codon) by using an analysis software for a nucleic acid and amino acid sequence (Genetyx, software development, Tokyo) as described in EXAMPLE 3 hereinafter. Further, as shown in SEQ ID NO: 2 for instance, a protein of the invention in which an amino acid sequence thereof is once determined can be prepared based on the sequence according to a method known to those skilled in the art for example, a method where each amino acid is polymerized chemically to synthesize a protein peptide synthesis method) Still further, in the case where a protein of the invention is encoded by the gene of the invention in the above, a protein of the invention can be obtained for example by producing a recombinant vector including an isolated gene of the invention to introduce into a proper host cell, followed by culturing or raising the resulting transformant in a medium to recover the protein from the culture or the resultant. A recombinant vector, host cell, medium, each procedure and conditions used herein can be selected appropriately from those known to people skilled in the art.

[0040] “Deletions, substitutions or additions of one or a plurality of amino acids” in the amino acid sequence of SEQ ID NO: 2 can be performed according to common techniques used at the time when this application is fled, for example, site-directed mutagenesis method (Zoller et al., Nucleic Acids Res. 10 6478-6500, 1982) by means of modifying a sequence of a gene (e.g. a nucleotide sequence of SEQ ID NO: 1) which encodes a protein comprising the amino acid sequence of SEQ ID NO: 2.

[0041] In this connection, terms “one or a plurality of amino acids” and “a protein having activity which alters the degree of dormancy of wheat seed” used herein have the same meaning as is described in the section of an isolated gene of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Embodiments of the invention will be further described below by means of illustration, but it is not intended that the scope of the invention is limited to them.

EXAMPLE 1

[0043] The Confirmation of Seed Dormancy

[0044] Seeds obtained from 4 varieties of wheat (Zenkoujikomugi, Minaminokomugi, Sanin 1 and Tozan 18) and seeds artificially treated to decrease dormancy (Minaminokomugi, Sanin 1) were soaked individually in 5% (W/V) Benlate T (DuPont, USA) for 10 minutes, then sterilized by washing with water to be laid in petri dish. 8 ml of distilled water was added to them respectively, followed by culturing at 22° C. for 5 days to determine a sprouting rate (the number of sprouting seeds in the total number of tested seeds is indicated by %).

[0045] The results are shown in Table 1 below. TABLE 1 The correlation between dormancy and sprouting rate of wheat Minaminokomugi (the Sanin 1 dormancy has been (the dormancy has Zenkbujikomugi Minaminokomugi Sanin 1 disrupted) been disrupted) Tozan 18 Dormancy strong strong weak — — weak Sprouting rate (%) 30 0 70 100 100 100 (22 ° C., 5 days)

[0046] As seen from the results of table 1, a test sample of wheat “Zenkoujikomugi” and “Minaminokomugi” which has been said to have strong dormancy (resistance to preharvest sprouting) in general exhibits a low sprouting rate of 30% and 0% respectively, and a test sample of wheat; “Sanin 1” and “Tozan 18” which has been said to have weak dormancy in general exhibits a high sprouting rate of 70% and 100%, respectively. Accordingly, a wheat seed sample of each variety used in the examples hereinafter was confirmed to have natural dormancy. Additionally, both of the test samples, “Minaminokomugi” and “Sanin 1” which are artificially treated to decrease dormancy exhibited an extremely high sprouting rate of 100%, which confirmed that they had been correctly treated to disrupt dormancy as in the above.

EXAMPLE 2

[0047] Analysis of Abscisic Acid-Sensitivity of a Seed Embryo in Each Variety

[0048] Next, seeds obtained from 3 varieties Minaminokomugi, Sanin 1 and Tozan 18) and seeds in which dormancy has been disrupted (Minaminokomugi, Sanin 1) were soaked individually in 5% (W/V) Benlate T(DuPont, USA) for 10 minutes, then sterilized by washing with distilled water. The grains of these wheat seeds were cut in halves to be laid in pet dish. A section where 8 mL of abscisic acid (ABA) solution was added and a section where 8 mL of distilled water was added were produced for each seed, followed by culturing at 22° C. for 6 days to determine the sprouting rate and the degree of sprout stretching.

[0049] The results of seeds of varieties having strong/weak dormancy and seeds in which dormancy has been disrupted by long-term storage are shown in Table 2 below, TABLE 2 Fluctuation of the sprouting rate and the degree of sprout stretching in 5 kinds of wheat by the abscisic acid treatment Minaminokomugi Sanin 1 (the dormancy has (the dormancy has Minaminokomugi Sanin 1 Tozan 18 been disrupted been disrupted Dormancy of seeds strong weak weak — — Seeds cut Sprouting + 10 μM 0 100 100 70 100 in halves Rate (%) ABA H₂O 90 90 100 90 90 Length of + 10 μM 0 2.96 4.65 2.37 4.50 a ABA (0.20) (0.80) (0.48) (0.53) sprout(m H₂O 56 73 64 52 74 m) (6.6) (1.9) (3.0) (6.5) (2.3) (Standard error)

[0050] As seen from the results of table 2, it was found that a seed of “Minaminokomugi” with strong dormancy had a strong abscisic acid-sensitivity, and seeds of “Sanin 1” and “Tozan 18” with weak dormancy, and seeds of “Minaminokomugi” and “Sanin 1” in which dormancy has been disrupted had weak abscisic acid-sensitivity or insensitivity. Taking into account the fact that a causal gene of mutation with abscisic acid-insensitivity in Arabidopsis was a homologous gene of field corn VP1 (described hereinbefore), a wheat of each variety, “Minaminokomugi” etc. is thought to have VP1 gene which affects the abscisic acid-sensitivity,

EXAMPLE 3

[0051] Decoding of a Nucleotide Sequence of cDNA Presumed to be Wheat VP1 Gene

[0052] First of all, whole RNA was isolated from a mature embryo of a seed of wheat variety “Minaminokomugi” using Trizole agent (Gibco Lifetech Oriental, U.S.A). mRNA was then extracted from the whole RNA using a Latex beads kit (Takara). Using the obtained mRNA as a template, cDNA was prepared with Poly T (Gibco Lifetech Oriental, U.S.A.) as primers using reverse transcriptase and a cDNA synthesis kit (both products were made by Gibco Lifetech Oriental, U.S.A.).

[0053] EcoRI adaptor (Gibco Lifetech Oriental, U.S.A.) was then added to the 5′ end and 3′ end of said cDNA respectively, followed by subcloning in a plasmid vector pBluescript IISK+(Staratagene), thereby producing a cDNA library of a mature wheat embryo.

[0054] Furthermore, by using a sequence known to be specifically preserved in a sequence of corn VP1 gene as primers, PCR was performed with LA PCR kit (Takara) to obtain a full-length clone of a gene presumed to be wheat VP1 gene from said cDNA library. To be more specific, first of all, 1 μL of cDNA sample solution containing the fill-length clone (2.5 ngDNA/μL), 5 μL of 10×LA PCR buffer (Mg 25 mM). 8 μL of dNTP mixture (2.5 mM each), 0.5 μL of forward primer (20 pmol/μL), 0.5 μL of reverse primer (20 pmol/μL), 0.5 μL of Takara LA Taq and 34.5 μL of sterilized water were poured into a micro tube with capacities of 0.5 mL, and they were mixed to prepare a reaction solution. The solution was then set in GeneAmp PCR system 9700 (PE Applied Biosystems, USA), and 30 cycles with each temperature profile of 30 sec at 96° C. 30 sec at 60 and 150 sec at 72 were performed to amplify DNA of a putative gene of wheat VP1. Sequences of primers used were as follows. (Primer) Forward primer: 5′-TGCATGGACGCCTCCGCCGG-3′ (SEQ ID NO:3) Reverse primer: 5′-TCAGATGCTCAC(G/C)GCCATCT-3′ (SEQ ID NO:4)

[0055] The amplified DNA was cloned in pT7 Blue T Plasmid Novagene, U.S.A.).

[0056] A nucleotide sequence of a plasmid which contains desired DNA fragment was then determined based on dideoxy method using Dye Terminator Cycle Sequencing FS Ready Reaction kit (PE Biosystems Japan, Mode1310 type). The obtained nucleotide sequence is represented by SEQ ID NO: 1. This nucleotide sequence was further analyzed using an analysis software for a nucleic acid and amino acid sequence (Genetyx, software development Tokyo). A sequence of 5′ side and 3′ side were determined by performing 5′ RACE and 3′ RACE respectively with an obtained internal sequence.

[0057] From the result, a gene of the invention is thought to consist of 2061 bases (SEQ ID NO: 1), encoding a protein comprising 687 amino acids (the amino acid sequence is shown in SEQ ID NO: 2).

[0058] Furthermore, the obtained amino acid sequence (SEQ ID NO: 2) was compared with a putative amino acid sequence from a nucleotide sequence of VP1 gene which is known in relation to different types of plants (cat grass, rice and field corn). The results are shown in FIG. 1.

[0059] From the results indicated in FIG. 1, the amino acid sequence (SEQ ID NO: 2) encoded by a gene of the invention was confirmed to show 66.6%, 60.5%, 58.6% homology to the sequences encoded by VP1 gene of cat grass, that of rice, and that of field corn, respectively. These values are extremely high, which indicates that they were encoded by homologous genes although their sequences differs to some extent.

EXAMPLE 4

[0060] Elucidation of the Correlation Between the Expression Level and Seed Dormancy in a Seed Embryo of a Gene Presumed to be Wheat VP 1 Gene.

[0061] Whole RNA was isolated form wheat seed samples of the same varieties (Zenkoujikomugi, Minaminokomugi, Sanin 1 and Tozan 18) as used in Example 1 and 2 using Trizole agent (Gibco Lifetech Oriental, USA). Then, only mRNA was extracted from the whole RNA using the Latex beads kit (TAKARA). These mRNA were run on agarose gel electrophoresis respectively, followed by blotting to N plus membrane (Amersham pharmacia, Sweden). DNA of a gene of the invention obtained above was then labeled with Alfos Direct Labelling Detection Kit (Amersham Pharmacia, Sweden) to be used as a probe, thereby performing hybridization to mRNA from samples of each variety blotted on the membrane.

[0062] The expression level of a gene of the invention was determined by judging the degree of hybridization to DNA of the gene of the invention used as a probe from the strength of a detected label. The results are shown in FIG. 2.

[0063] As a result the correlation was confirmed between the degree of dormancy(strong/weak) and the expression level of said gene. That is to say, the expression level of said gene was high in a seed embryo of varieties with strong dormancy, “Minaminokomugi” and “Zenkoujikomugi” while the expression level of said gene was low in a seed embryo of varieties with weak dormancy, “Sanin 1” and “Tozan 18”, and in seed embryos in which dormancy has been disrupted, “Minaminokomugi” and “Sanin 1”.

[0064] Considering the above results and the results obtained from Example 1 and 2 together, said gene presumed to be wheat VP1 gene is considered to alter the degree of abscisic acid-sensitivity of a seed depending on the difference of the expression level between varieties, thereby changing the degree of dormancy of said seed.

Effect of the Invention

[0065] The present invention provides a gene which regulates the dormancy of wheat seed, an antisense DNA to said gene, and a protein having activity which alters the degree of dormancy of wheat seed.

Free Text of Sequence Listing

[0066] SEQ ID NO: 2: a putative amino acid sequence from a nucleotide sequence of SEQ ID NO: 1.

[0067] SEQ ID NO: 3: a forward primer used to amplify a sequence of the gene of the invention

[0068] SEQ ID NO: 4: a reverse primer used to amplify a sequence of the gene of the invention.

[0069] All publications, oatents and patent applications cited herein are incorporated herein by reference in their entirety.

1 4 1 2061 DNA Triticum aestivum L. 1 atggacgcct ccgccggctc gtcgccgccg cggcactcac agggggacgc gccgaggcgc 60 ggcgggccgc accgcgggaa gggccccgcg gtggagatcc ggcagggaga ggacgacttc 120 atgttcgcgc aggatacctt cccggccctc ccggacttcc cttgcctctc ctcgccgtcg 180 agctccacct tctcctcctc ctcgtcttcc aactcctcca gcgccttcgc tgccccagcg 240 ggagcgggcg ggcgcggggc cgagggggcg cgcggcgagc cgtccgagcc tgcagcggcc 300 ggggacggga tggacgacct ctccgacatc gaccacctgc tcgacttcgc gtccatcaac 360 gacgacgtcc cctgggacga cgagccgctc ttccccgacg tcgggatgat gctggaggat 420 gtcatctccg agcagcagct ccagcagcct ccggcgggcc acggcacggc cgggagaacg 480 gcgccgcatg cggcctctgg tggaggagag gatgccttca tgggtggcgg cggcacgggg 540 agcgcggcgg acgacctgcc gctgttcttc atggagtggc tcaagaacaa ccgcgactgc 600 atctcggccg aggacctccg cagcatccgc ctccgtcgat ccaccatcga ggccgcggcc 660 gcgcgcctcg gtggggggcg ccagggcacc atgcagctgc tcaagctcat cctcacctgg 720 gtgcagaacc accacctgca gaagaagcgc ccccgcgtcg gcgccatgga tcaggaggcg 780 ccgccggcag gaggccagct ccctagcccc ggcgcaaacc ccggctacga attccccgcg 840 gagacgggtg ccgccgctgc cacatcatgg atgccctacc aggccttctc gccaactgga 900 tcctacggcg gcggggcgat ctacccattc cagcagggct gcagcactag cagcgtggcc 960 gtgagcagcc agccgttctc cccgccggcg gcgcccgaca tgcacgccgg ggcctggccc 1020 ctgcagtacg cggcgttcgt cccagctgga gccacatccg caggcactca aacatacccg 1080 atgccgccgc cgggggccgt gccgcagccg ttcgcggccc ccggattcgc cgggcagttc 1140 ccgcagcgga tggagccggc ggcgaccagg gaggcccgga agaagcggat ggcgaggcag 1200 cggcgcttgt cgtgcctgca gcagcagcgg agccagcagc tgaatctgag ccagatccaa 1260 accggcggct tccctcaaga gcaatccccc cgcgcggcgc actcggcgcc ggtcacgccg 1320 ccgtcgtccg gctggggagg cctctgggcg caacaggccg tccagggcca gctcatggtc 1380 caggtcccga atccgctgtc gacgaagtcc aattcctcaa ggcagaagca gcaaaaaccc 1440 tcgccggacg cagcagcgag gccgccctcc ggcggcgccg ccacgccgca tcgcccgggg 1500 caggcgtcgg cttccaataa gcagcggcag cagggtgcga ggacgccggc ggcggcgccg 1560 gcggcaggag acaagaacct gcggttcctg ctgcagaagg tgctcaagca gagcgacgtc 1620 ggaaccctcg gccgcatcgt gctccccaaa gaagcggaga ctcacctgcc ggagctcaag 1680 acgggggacg gcatctcgat ccccattgag gacatcggca catctcaggt ctggagcatg 1740 cggtaccgat tttggcccaa caacaagagc agaatgtatc ttctagagaa cactggtgac 1800 tttgttcggt ccaatgagct gcaggagggt gatttcatcg tgctttactc tgatgtcaag 1860 tcaggcaaat atctgatacg cggcgtgaag gtaagagctc aacaggatct agccaagcac 1920 aagaatgcca gtccagagaa aggtggggcg tccgacgtga aggcgggcgg agaagacggt 1980 ggttgcaaag agaagtctcc gcacggtgtc cggcgatctc gccaggaagc cagctccatg 2040 aaccagatgg ccgtgagcat c 2061 2 687 PRT Artificial Sequence Description of Artificial Sequence Polypeptide sequence deduced from the polynucleotide sequence of SEQ ID No1. 2 Met Asp Ala Ser Ala Gly Ser Ser Pro Pro Arg His Ser Gln Gly Asp 1 5 10 15 Ala Pro Arg Arg Gly Gly Pro His Arg Gly Lys Gly Pro Ala Val Glu 20 25 30 Ile Arg Gln Gly Glu Asp Asp Phe Met Phe Ala Gln Asp Thr Phe Pro 35 40 45 Ala Leu Pro Asp Phe Pro Cys Leu Ser Ser Pro Ser Ser Ser Thr Phe 50 55 60 Ser Ser Ser Ser Ser Ser Asn Ser Ser Ser Ala Phe Ala Ala Pro Ala 65 70 75 80 Gly Ala Gly Gly Arg Gly Ala Glu Gly Ala Arg Gly Glu Pro Ser Glu 85 90 95 Pro Ala Ala Ala Gly Asp Gly Met Asp Asp Leu Ser Asp Ile Asp His 100 105 110 Leu Leu Asp Phe Ala Ser Ile Asn Asp Asp Val Pro Trp Asp Asp Glu 115 120 125 Pro Leu Phe Pro Asp Val Gly Met Met Leu Glu Asp Val Ile Ser Glu 130 135 140 Gln Gln Leu Gln Gln Pro Pro Ala Gly His Gly Thr Ala Gly Arg Thr 145 150 155 160 Ala Pro His Ala Ala Ser Gly Gly Gly Glu Asp Ala Phe Met Gly Gly 165 170 175 Gly Gly Thr Gly Ser Ala Ala Asp Asp Leu Pro Leu Phe Phe Met Glu 180 185 190 Trp Leu Lys Asn Asn Arg Asp Cys Ile Ser Ala Glu Asp Leu Arg Ser 195 200 205 Ile Arg Leu Arg Arg Ser Thr Ile Glu Ala Ala Ala Ala Arg Leu Gly 210 215 220 Gly Gly Arg Gln Gly Thr Met Gln Leu Leu Lys Leu Ile Leu Thr Trp 225 230 235 240 Val Gln Asn His His Leu Gln Lys Lys Arg Pro Arg Val Gly Ala Met 245 250 255 Asp Gln Glu Ala Pro Pro Ala Gly Gly Gln Leu Pro Ser Pro Gly Ala 260 265 270 Asn Pro Gly Tyr Glu Phe Pro Ala Glu Thr Gly Ala Ala Ala Ala Thr 275 280 285 Ser Trp Met Pro Tyr Gln Ala Phe Ser Pro Thr Gly Ser Tyr Gly Gly 290 295 300 Gly Ala Ile Tyr Pro Phe Gln Gln Gly Cys Ser Thr Ser Ser Val Ala 305 310 315 320 Val Ser Ser Gln Pro Phe Ser Pro Pro Ala Ala Pro Asp Met His Ala 325 330 335 Gly Ala Trp Pro Leu Gln Tyr Ala Ala Phe Val Pro Ala Gly Ala Thr 340 345 350 Ser Ala Gly Thr Gln Thr Tyr Pro Met Pro Pro Pro Gly Ala Val Pro 355 360 365 Gln Pro Phe Ala Ala Pro Gly Phe Ala Gly Gln Phe Pro Gln Arg Met 370 375 380 Glu Pro Ala Ala Thr Arg Glu Ala Arg Lys Lys Arg Met Ala Arg Gln 385 390 395 400 Arg Arg Leu Ser Cys Leu Gln Gln Gln Arg Ser Gln Gln Leu Asn Leu 405 410 415 Ser Gln Ile Gln Thr Gly Gly Phe Pro Gln Glu Gln Ser Pro Arg Ala 420 425 430 Ala His Ser Ala Pro Val Thr Pro Pro Ser Ser Gly Trp Gly Gly Leu 435 440 445 Trp Ala Gln Gln Ala Val Gln Gly Gln Leu Met Val Gln Val Pro Asn 450 455 460 Pro Leu Ser Thr Lys Ser Asn Ser Ser Arg Gln Lys Gln Gln Lys Pro 465 470 475 480 Ser Pro Asp Ala Ala Ala Arg Pro Pro Ser Gly Gly Ala Ala Thr Pro 485 490 495 His Arg Pro Gly Gln Ala Ser Ala Ser Asn Lys Gln Arg Gln Gln Gly 500 505 510 Ala Arg Thr Pro Ala Ala Ala Pro Ala Ala Gly Asp Lys Asn Leu Arg 515 520 525 Phe Leu Leu Gln Lys Val Leu Lys Gln Ser Asp Val Gly Thr Leu Gly 530 535 540 Arg Ile Val Leu Pro Lys Glu Ala Glu Thr His Leu Pro Glu Leu Lys 545 550 555 560 Thr Gly Asp Gly Ile Ser Ile Pro Ile Glu Asp Ile Gly Thr Ser Gln 565 570 575 Val Trp Ser Met Arg Tyr Arg Phe Trp Pro Asn Asn Lys Ser Arg Met 580 585 590 Tyr Leu Leu Glu Asn Thr Gly Asp Phe Val Arg Ser Asn Glu Leu Gln 595 600 605 Glu Gly Asp Phe Ile Val Leu Tyr Ser Asp Val Lys Ser Gly Lys Tyr 610 615 620 Leu Ile Arg Gly Val Lys Val Arg Ala Gln Gln Asp Leu Ala Lys His 625 630 635 640 Lys Asn Ala Ser Pro Glu Lys Gly Gly Ala Ser Asp Val Lys Ala Gly 645 650 655 Gly Glu Asp Gly Gly Cys Lys Glu Lys Ser Pro His Gly Val Arg Arg 660 665 670 Ser Arg Gln Glu Ala Ser Ser Met Asn Gln Met Ala Val Ser Ile 675 680 685 3 20 DNA Artificial Sequence Description of Artificial Sequence Forward primer used to amplify the sequence of the gene of the invention. 3 tgcatggacg cctccgccgg 20 4 20 DNA Artificial Sequence Description of Artificial Sequence Reverse primer used to amplify the sequence of the gene of the invention. 4 tcagatgctc acsgccatct 20 

1. An isolated gene encoding the amino acid sequence of SEQ ID NO:
 2. 2. An antisense DNA to the gene according to claim
 1. 3. An isolated protein comprising the amino acid sequence of SEQ ID NO:
 2. 4. A recombinant vector comprising the gene according to claim
 1. 5. A recombinant vector comprising the antisense DNA according to claim
 2. 6. A transformant comprising the recombinant vector according to claim
 4. 7. A transformant comprising the recombinant vector according to claim
 5. 8. A method for producing a protein having activity which alters the degree of dormancy of wheat seed comprising culturing the transformant according claim 6 and recovering the protein from the resultant culture.
 9. A method for producing a protein having activity which alters the degree of dormancy of wheat seed comprising culturing the transformant according claim 7 and recovering the protein from the resultant culture. 